Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of controlling insect pests. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Coleopterans are a significant group of agricultural pests that cause extensive damage to crops each year. Examples of coleopteran pests include but are not limited to: Colorado potato beetle (CPB), corn rootworm, alfalfa weevil, boll weevil, and Japanese beetle. The Colorado potato beetle is an economically important pest that feeds on the leaves of potato, eggplant, tomato, pepper, tobacco, and other plants in the nightshade family. The Colorado potato beetle is a problematic defoliator of potatoes, in part, because it has developed resistance to many classes of insecticides.
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is one of the most devastating coleopterans in North America and is a particular concern in corn-growing areas of the Midwestern United States. Approximately 9.3 million acres of U.S. corn are infested with corn rootworm species complex each year. The northern corn rootworm (NCR), Diabrotica barberi Smith and Lawrence, is a closely-related species that co-inhabits much of the same range as WCR. There are several other related subspecies of Diabrotica that are significant pests in the Americas: the Mexican corn rootworm (MCR), D. virgifera zeae Krysan and Smith; the southern corn rootworm (SCR), D. undecimpunctata howardi Barber; D. balteata LeConte; D. undecimpunctata tenella; D. speciosa Germar; and D. u. undecimpunctata Mannerheim. The United States Department of Agriculture has estimated that corn rootworms cause $1 billion in lost revenue each year, including 800 million in yield loss and 200 million in treatment costs.
Both WCR and NCR eggs are deposited in the soil during the summer. The insects remain in the egg stage throughout the winter. The larvae hatch in late May or early June and begin to feed on corn roots. Corn rootworms go through three larval instars. After feeding for several weeks, the larvae molt into the pupal stage. They pupate in the soil, and then emerge from the soil as adults in July and August.
Most of the rootworm damage in corn is caused by larval feeding. Newly hatched rootworms initially feed on fine corn root hairs and burrow into root tips. As the larvae grow larger, they feed on and burrow into primary roots. When corn rootworms are abundant, larval feeding often results in the pruning of roots all the way to the base of the corn stalk. Severe root injury interferes with the roots ability to transport water and nutrients into the plant, reduces plant growth, and results in reduced grain production, thereby often drastically reducing overall yield. Severe root injury also often results in lodging of corn plants, which makes harvest more difficult and further decreases yield. Furthermore, feeding by adults on the corn reproductive tissues can result in pruning of silks at the ear tip. If this “silk clipping” is severe enough during pollen shed, pollination may be disrupted. In addition, members of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.
Control of corn rootworms has been attempted by crop rotation, chemical insecticides, biopesticides such as the spore-forming gram-positive bacterium, Bacillus thuringiensis (B.t.), transgenic plants that express B.t. toxins, and a combination thereof. Crop rotation suffers from the disadvantage of placing unwanted restrictions upon the use of farmland. Moreover, oviposition of some rootworm species may occur in soybean fields, thereby compromising the effectiveness of crop rotation practiced with corn and soybean.
Chemical insecticides are the most heavily relied upon strategy for achieving corn rootworm control. Chemical insecticide use is an imperfect corn rootworm control strategy; high populations of larvae, heavy rains, and improper application of the insecticide(s) may all result in inadequate corn rootworm control. Furthermore, the continual use of insecticides may select for insecticide-resistant rootworm strains, as well as raise significant environmental concerns due to their toxicity to non-target species.
Damage to plants caused by nematodes is also a prevalent and serious economic problem. Nematodes cause wide-spread and serious damage in many plant species. Many genera of nematodes are known to cause such damage. Plant-parasitic nematodes include members of the Phylum Nematoda, Orders Tylenchida and Dorylaimide. In the Order Tylenchida, the plant-parasitic nematodes are found in two Super Families: Tylenchoidea and Criconematoidea. There are more than 100,000 described species of nematodes.
Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals that might be found in food, ground water, and the environment. Stringent new restrictions on the use of chemical pesticides and the elimination of some effective pesticides from the marketplace could limit economical and effective options for controlling costly pests. Thus, there is an urgent need to identify new pest control agents and compositions.
Regular use of chemical pesticides for the control of unwanted insect pests can select for chemical resistant strains. Chemical resistance occurs in many species of economically important insects and has also occurred in nematodes of sheep, goats, and horses. For example, an accepted methodology for control of nematodes has centered around the drug benzimidazole and its congeners. The use of these drugs on a wide scale has led to many instances of resistance among nematode populations (Prichard, R. K. et al). The development of pesticide resistance necessitates a continuing search for new control agents having different modes of action.
At the present time there is a need to have more effective means to control the many coleopterans and nematodes that cause considerable damage to susceptible hosts and crops. Advantageously, such effective means would employ highly selective biological toxins. Several B.t. Cry proteins have been shown to be nematicidal, these include Cry5B, Cry6A, Cry14A and Cry21A (Wei et al., 2003; Aroian and Li (2010).
B.t. is a soil-borne bacterium that produces pesticidal crystal proteins known as delta endotoxins or Cry proteins. Cry proteins are oral intoxicants that function by acting on midgut cells of susceptible insects. Some Cry toxins have been shown to have activity against nematodes. An extensive list of delta endotoxins is maintained and regularly updated at the Bacillus thuringiensis Toxin Nomenclature web site maintained by Neil Crickmore. (See www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/toxins2.html and Crickmore et al. 1998, page 808). Cry toxins, including members of the Cry 1B, Cry 1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C (Frankenhuyzen, 2009) families have insecticidal activity against coleopteran insects.
Some B.t. toxins which are active against corn rootworm and other coleopterans are now known. Cry6Aa has reported activity against coleopteran and nematode pests (U.S. Pat. Nos. 5,186,934; 6,632,792 B2; U.S. Pat. No. 2011/0225681; U.S. Pat. No. 2011/0239334 A1; and Wei et al., 2003). For example, U.S. Pat. No. 4,849,217 discloses various isolates, including PS52A1 and PS86A1, as having activity against alfalfa weevils. U.S. Pat. No. 5,208,017 discloses PS86A1 as a having activity against Western corn rootworm. U.S. Pat. Nos. 5,427,786 and 5,186,934 each disclose B.t. isolates and toxins active against coleopterans. Specifically disclosed in these patents is the isolate known as PS86A1 and a coleopteran-active toxin obtainable therefrom known as 86A1. Toxin 86A1 is now also known as Cry6A (CryVIA). The wild-type Cry6Aa toxin is about 54.1 kDa. A Cry6B toxin is also known. This toxin can be obtained from the PS69D1 isolate. Cry6Aa is recognized as a new mode of action against western corn rootworm, complementing Cry3Aa and Cry34Ab1/Cry35Ab1 (Li et al, 2013) making it a pyramid partner in an integrated insect resistance management program (U.S. Pat. No. 2013/0167269 A1 and US 2013/0263331 A1).
The full length Cry6A and Cry6B toxins are known to have activity against nematodes. The PS69D1 isolate has been reported to have activity against nematodes (U.S. Pat. Nos. 4,948,734; 5,093,120; 5,262,399; and 5,439,881). A generic formula for the amino acid sequence of CryVI toxins has been disclosed in WO 92/19739, which also teaches that the full length toxin has activity against nematodes. The PS52A1 and PS69D1 isolates are disclosed therein. U.S. Pat. Nos. 5,262,159 and 5,468,636 also disclose a generic formula for toxins having activity against aphids.
Cry6A toxin is known to inhibit the growth of certain coleopterans and can be activated by enzymatically cleaving to yield an amino terminal core toxin that is lethal to coleopterans, such as the western corn rootworm (U.S. Pat. No. 6,831,062 B2). In addition, truncated Cry6A is active against nematodes. U.S. Pat. No. 6,831,062 describes Cry6A truncated holotoxins and fusion proteins and fusion genes. Thompson et al disclosed the insecticidally active peptide fragments identified as being residues 12-390 and 12-443 depending on the cleavage site. The large fragment, from approximately residues 12-390 or 12-443 resulting from trypsin, or other proteolytic digestion, are called the core fragments or toxins. The trypsin treatment of Cry6Aa, produced from recombinant B.t., increased activity against WCR (U.S. Pat. Nos. 5,874,288; 6,831,062 B2; and 6,303,364 B1).
Although production of the currently-deployed Cry proteins in transgenic plants can provide robust protection against the aforementioned pests, thereby protecting grain yield, adult pests have emerged in artificial infestation trials, indicating less than complete larval insect control. Additionally, development of resistant insect populations threatens the long-term durability of Cry proteins in insect pest control. Coleopteran insects have developed resistance in the field to Cry proteins (Gassman et al. PLoS ONE July 2011|Volume 6|Issue 7|e22629). Insect resistance to B.t. Cry proteins can develop through several mechanisms (Heckel et al., 2007; Pigott and Ellar, 2007). Multiple receptor protein classes for Cry proteins have been identified within insects, and multiple examples exist within each receptor class. Resistance to a particular Cry protein may develop, for example, by means of a mutation within the toxin-binding portion of a cadherin domain of a receptor protein. A further means of resistance may be mediated through a protoxin-processing protease.
While native Cry6Aa naturally expressed in B.t. strains has shown good efficacy against WCR and certain nematodes, its use as an effective plant incorporated protectant has not been demonstrated due to its susceptibility to proteolysis when expressed in plant cells. Therefore, engineering Cry6A toxins to be more resistant to proteolysis when expressed in plant cells would be highly desirable for use in recombinant plants, especially corn, as a plant-incorporated protectant.